JP5174457B2 - Neisseria meningitidis lgtBLOS as an adjuvant - Google Patents

Abstract

The present invention relates to Neisserial Lipo-Oligo-Saccharides (LOS) that comprise a tri-saccharide outer core that shows increased binding to the DC-SIGN receptor on dendritic cells, as a result of which the Neisserial LOS's of the invention have increased immunostimulatory activity. The tri-saccharide outer core of the Neiserial LOS's combined with a Lipid A moiety with reduced toxicity is useful as an adjuvant in vaccine preparations.

Description

  The present invention relates to Neisserial lipooligosaccharides (LOS) with enhanced immunostimulatory activity. The Neisseria LOS of the present invention comprises a trisaccharide outer core that exhibits enhanced binding to receptors on dendritic cells. The combination of the Neisseria sp. LOS trisaccharide outer core and reduced toxicity Lipid A moiety is useful as an adjuvant in vaccine formulations.

  Neisseria meningitidis LPS is attracting attention as a potential vaccine candidate not only because of its potent adjuvant, but also because of its ability to induce an LPS-specific immune response. However, due to the endotoxin activity of LPS, its use in vaccines is limited so far.

  Meningococcal LPS consists of a hydrophobic lipid A moiety that anchors itself to the bacterial outer membrane and a hydrophilic oligosaccharide core that is exposed on the bacterial surface. (FIG. 1). Since natural oligosaccharide chains are structurally similar to host glycolipid antigens, it is undesirable to include them in vaccine formulations. Gene recombination of the oligosaccharide biosynthetic pathway of LPS makes it possible to produce a series of meningococcal mutants that express truncated oligosaccharide chains (FIG. 1). The endotoxin and adjuvant properties of LPS are thought to be determined by the lipid A portion of such molecules. The inventors have previously created a unique set of meningococcal lipid A mutants with greatly improved pharmacological properties. Specifically, when the lpxL1 (htrB1) gene associated with acyloxyacylation of lipid A was inactivated, a lipid A mutant strain expressing pentaacylated lipid A, but not hexaacylated lipid A, could be isolated. Such mutant strains maintained the adjuvant activity while reducing the endotoxin activity. In addition, a Neisseria meningitidis mutant completely lacking LPS was also isolated by inactivation of the Neisseria meningitidis lpxA gene. Thus, not only the oligosaccharide part of the meningococcal LPS but also the modification of the lipid A part has opened up new possibilities in the development of a meningococcal vaccine.

  Dendritic cells (DC) are of great interest because they play a role in initiating both the immune response in natural infections and the immune response to vaccination. The immune response against bacteria is initiated by DC. DCs phagocytose and process bacterial antigens and present them to T cells. We have recently shown that meningococcal LPS plays an important role during interaction with human DCs. In addition, LPS is also required for bacterial internalization and complete activation of DCs.

Definitions As used herein, an adjuvant, when used in combination with an antigen, immunizes a mammal, preferably a human, and stimulates the immune system, thereby preferably producing no specific immune response against the adjuvant itself, Defined as including any substance or compound that elicits, enhances or promotes an immune response to said antigen. Suitable adjuvants enhance the immune response against a given antigen, at least 1.5, 2, 2.5 compared to the immune response generated against said antigen under similar conditions but without the adjuvant. Enhances immune response to 5, 10, or 20 times stronger. In the art, analytical methods can be used that determine the statistical mean enhancement value of the immune response to a given antigen produced in an animal or human population by an adjuvant in the context of a corresponding control population. Said adjuvant is preferably capable of enhancing the immune response to at least two different antigens. The adjuvants of the present invention are usually compounds that are foreign to the mammal, thus excluding immunostimulatory compounds that are endogenous to the mammal, such as interleukins, interferons, and other hormones.

  The present invention is based on the surprising discovery that Neisseria LPS having a specific trisaccharide outer core structure enhances binding and internalisation to human dendritic cells (DC). Binding and internalization of LPS containing trisaccharide, more precisely LOS (lipo-oligosaccharide) containing trisaccharide is mediated by C-type lectin DC-SIGN expressed in human DC, and activation and maturation of DC Increase the speed of. This novel trisaccharide-containing Neisseria sp. LOS can be used as a powerful adjuvant in immunization and vaccination due to its improved immunostimulatory action.

で表され、式中、ＬＡが、好ましくはナイセイア属菌株由来の野生型リピドＡ部分、又はより好ましくは毒性が低下したリピドＡ部分であることを特徴とする方法に関する。 That is, the first aspect of the present invention is a method of immunizing or vaccinating a mammal with an antigen, the method comprising administering the antigen (to the mammal), and Neisseria spp. Formula (I)

Wherein LA is preferably a wild-type lipid A moiety from a Neisseria strain, or more preferably a lipid A moiety with reduced toxicity.

  In the formula (1), GlcNAc is D-glucosamine, Gal is D-galactose, Glc is D-glucose, Hep is D-heptose, KDO is 3-deoxy-D-mannococtulosonic acid, PEA is phosphoethanolamine, β1 → 4 is a β-glycoside bond between the 1st and 4th positions, β1 → 3 is a β-glycoside bond between the 1st and 3rd positions, and α1 → 5 is a position between the 1st and 5th positions. Α-glycoside bond, α1 → 3 is the α-glycoside bond between the 1st and 3rd positions, α1 → 2 is the α-glycoside bond between the 1st and 2nd positions, and α2 → 4 is It is an α-glycoside bond between the 2 and 4 positions, and also PEA (3 or 6) may have phosphoethanolamine bonded at the 3 or 6 position of heptose or not present at all It shows that it may be.

  The invention thus relates to the use of Neisseria LOS as defined above in the manufacture of a medicament for immunization or vaccination of mammals. The Neisseria LOS of the present invention is preferably used as an adjuvant. Said Neisseria spp. LOS is more preferably used in combination with an antigen in the manufacture of a medicament for eliciting an immune response to an antigen (or vaccination with an antigen) in a mammal.

  In the methods and uses of the present invention, the mammal is preferably a human and the antigen is preferably derived from a bacterium, virus, fungus, parasite, cancer cell, or allergen as defined below, Or produced from them. The antigen and Neisseria LOS may be used for the treatment and / or prevention of infections caused by bacteria, viruses, fungi or parasites, tumors caused by cancer cells, or allergies caused by allergens. preferable.

  In a preferred embodiment of the invention, the Neisseria LOS of the invention has a modified lipid A moiety (LA) with reduced toxicity. The toxicity of such modified LA is preferably lower than that of the corresponding wild type LA, more preferably 90, 80, 60, 40, 20, 10, 5, 2, 1, 0 of the wild type LA toxicity. .5 or less than 0.2%. The toxicity of the wild type and various modified LAs with reduced toxicity can be determined using appropriate assays known in the art. A suitable assay for determining said toxicity, ie the biological activity of the LA or Neisseria LOS of the present invention, is the WEHI test examining TNF-alpha induction in the MM6 macrophage cell line (Espevik and Niessen, 1986, J. Immunol. Methods 95: 99-105; Ziegler-Heitbrock et al., 1988, Int. J. Cancer 41: 456-461).

  The Neisseria LOS of the present invention having LA with reduced toxicity, on the other hand, maintains sufficient immunostimulatory activity, ie, adjuvant activity. The Neisseria spp. LOS of the present invention with reduced toxicity preferably has at least 10, 20, 40, 80, 90, or 100% immunostimulatory activity of the corresponding Neisseria spp. LOS with wild-type LA. Such immunostimulatory activity is determined by measuring the production of at least one cytokine or the expression of at least one costimulatory molecule as described in Example 3 herein.

  Neisseria LOS having a lipid A portion with reduced toxicity but maintaining (partially) adjuvant properties can be obtained from genetically modified Gram-negative pathogens outlined in, for example, WO 02/09746. Specifically, preferred examples of Neisseria spp. LOS having LA with reduced toxicity include (a) 1 or 2 of the lpxL1 and lpxL2 genes (previously called htrB and msbB genes) or homologues thereof. LOS having a lipid A moiety that can be obtained from a Neisseria strain having a mutation that reduces or inactivates the above expression (see, for example, EP1,127,137 and US5,997,881), (b) acyloxyhydrolase ( LOS having a lipid A moiety that can be obtained by removing one or more secondary O-linked esterified fatty acids from a wild-type lipid A moiety by treatment with acyloxyhydrolase) or alkaline hydrolysis treatment (US 5, US Pat. 103,661 and Erwin et al., 1991, Infect, Immun, 59: 1881-87), (c) the wild type lipid A moiety LOS with a lipid A moiety that can be obtained by treatment with drazine (Gu et al., 1996, Infect. Immun. 64: 4047-53, Polotsky et al., 1994, Infect Immun, 62: 210-14, and Gu et al., 1998, Infect, Immun, 66: 1891-97, 1998), (d) LOS with a lipid A moiety that can be obtained by dephosphorylating the lipid A moiety using alkaline phosphatase (US6 290, 952), and (e) by enzymatically dephosphorylating or deacylating LOS produced in a Neisseria host expressing a heterologous lpxE gene or pagL gene. LOS that can be mentioned. As used herein, decreased expression is understood to mean an increase or decrease when compared to the corresponding wild-type strain, and the expression level is preferably at least 2-fold, 5-fold or 10-fold different from the corresponding wild-type strain. To do.

Thus, suitable LA toxicity is decreased, only secondary C ₁₂ to the reducing glucosamine _- LA having an acyl, non-reducing glucosamine only secondary C _{12 -} LA having an acyl, any secondary C _{12 -} acyl group LA that also lacks, LA that lacks one or more phosphate groups at either the 1 or 4 ′ position, and LA that has a combination of the above deacylation and dephosphorylation.

で表されるナイセリア属菌ＬＯＳであって、式中、好ましくはＬＡが、上記に定義した、毒性が低下したリピドＡ部分であることを特徴とするナイセリア属菌ＬＯＳに関する。 In a further aspect, the present invention provides a compound of formula (I)

The present invention relates to a Neisseria genus LOS represented by the formula, wherein LA is preferably a lipid A moiety with reduced toxicity as defined above.

Yet another aspect of the present invention relates to a composition comprising a Neisseria LOS as defined herein above and a pharmaceutically acceptable carrier. Preferably, the composition further comprises an antigen derived from or produced from bacteria, viruses, fungi, parasites, cancer cells, or allergens. The pharmaceutical composition may further include a pharmaceutically acceptable stabilizer, an osmotic agent, a buffer, a dispersant and the like. The preferred form of the pharmaceutical composition depends on the desired mode of administration and therapeutic application. The pharmaceutical carrier may be any compatible and non-toxic substance suitable for delivering active ingredients, namely Neisseria LOS and antigen, to a patient. Examples of pharmaceutically acceptable carriers for intranasal administration include water, buffered saline, glycerin, polysorbate 20, cremophor EL, mixed aqueous solution of caprylic / capric glycerides. And may be treated with a buffer to provide a neutral pH environment. Examples of pharmaceutically acceptable carriers for parenteral administration include 0.9% sodium chloride sterile buffer or 5% glucose buffer with 20% albumin added as needed. Preparations for parenteral administration must be sterile. Examples of parenteral routes of administration of an active ingredient by known methods include injection or infusion by subcutaneous, intravenous, intraperitoneal, intramuscular, intraarterial or intralesional routes. The composition of the present invention is preferably administered by bolus injection. A typical pharmaceutical composition for intramuscular injection is for example 1-10 ml of phosphate buffered saline, 1-100 μg, preferably 15-45 μg of antigen, and 1-100 μg, preferably 15-45 μg of the invention. Of Neisseria spp. LOS. In the case of oral administration, the active ingredient can be administered in the form of solutions such as elixirs, syrups, and suspensions. Liquid preparations for oral administration can contain coloring and flavoring to increase patient acceptance. Process for the preparation of parenteral, oral, or intranasal administration compositions are well known in the art, for example, ^{Remington's Pharmaceutical Science (15 th ed} ., Mack Publishing, Easton, PA, 1980) ( said by reference Details are described in various sources, including the entire literature).

  The antigen in the composition of the present invention is preferably an antigen derived from or produced from bacteria, viruses, fungi, parasites, cancer cells, or allergens. Viral antigens that can be combined with the Neisseria LOS of the present invention can be obtained from any virus. Examples of the virus include, but are not limited to, retroviridae such as human immunodeficiency virus (HIV), rubella virus, influenza virus, measles, mumps, respiratory syncytial virus, human metabolite Paramyxoviridae such as pneumonia virus and flavis such as yellow fever virus, dengue virus, hepatitis C virus (HCV), Japanese encephalitis virus (JEV), tick-borne encephalitis virus, St. Louis encephalitis virus or West Nile virus Viridae (flaviviridae), Herpesviridae (herpesviridae) such as herpes simplex virus, cytomegalovirus, Epstein-Barr virus, Bunyaviridae, Arenaviridae, Hantaviridae such as hunter, and coronavirus And papobau of human papilloma virus Irsidae, Rhabdoviridae such as rabies virus, Coronaviridae such as human coronavirus, Alphaviridae, Arteriviridae, Filoviridae such as Ebola virus, Arenavirus, and smallpox virus Poxviridae and African swine fever virus. Similarly, the Neisseria LOS of the present invention may be combined with antigens from pathogenic bacteria, fungi (including yeast), or parasites. Examples of the antigen include Helicobacter genus such as Helicobacter pylori, Neisseria genus such as Neisseria meningitidis, Haemophilus genus such as Haemophilus influenza, Bordetella genus such as Bordetella pertussis, Chlamydia genus, Streptococcus sp. bacterial antigens of Gram-negative enteric pathogens including Streptococcus genus such as serotype A), Vibrio genus such as Vibrio cholera, Salmonella genus, Shigella genus, Campylobacter genus and Escherichia genus, and anthrax, leprosy, tuberculosis , Antigens from causative bacteria of diphtheria, Lyme disease, syphilis, typhoid, and gonorrhea. Parasite-derived antigens include, for example, protozoa such as Babesis bovis, Plasmodium, Leishmania, Toxoplasma gondii, and Trypanosoma such as Cruise Trypanosoma. Examples of fungal antigens include antigens derived from fungi such as Aspergillus, Candida albicans, Cryptococcus genus such as Cryptococcus neoformans, and Histoplasma capsulatum.

  Vaccination is usually applied to preventive protection against pathogenic bacteria or treatment of diseases following pathogenic infections, but it is also well known to those skilled in the art that vaccines are applied to the treatment of tumors. In addition, many tumor-specific proteins have been found as suitable entities that can be targeted by human or humanized antibodies. Such tumor specific proteins are also included within the scope of the present invention. Many tumor-specific antigens are well known in the art. Accordingly, in one preferred embodiment, the present invention provides a composition comprising a tumor specific antigen and a Neisseria sp. LOS as defined above. Suitable tumor antigens include, for example, carcinoembryonic antigen, prostate specific membrane antigen, prostate specific antigen, MZ2-E protein, polymorphic epithelial mucin (PEM), folate binding protein LK26, truncated epidermis Growth factor receptor (EGRF), HER2, Thomasen-Friedenreich (T) antigen, GM-2 and GD2 ganglioside, Ep-CAM, mucin-1, epithelial glycoprotein-2, and colon specific antigen Can be mentioned.

  Furthermore, dendritic cells can be targeted by antigens in order to induce tolerance in the prevention of autoimmune diseases. Such allergens are also included within the scope of the present invention.

  Another aspect of the present invention relates to a method for producing the Neisseria LOS defined above. Preferred methods are: (a) expressing an immunotype L2, L3, or L4 Neisseria LOS and a meningococcal strain lacking an active lgtB gene, or an immunotype L6 Neisseria LOS Culturing a meningococcal strain additionally having a mutation that inactivates or reduces the expression of one or more lpxL1 gene or lpxL2 gene or homologues thereof, (b) Recovering Neisseria LOS. Alternatively, the method may comprise: (a) a Neisseria spp. LOS expressing an immunotype L2, L3, or L4 and lacking an active lgtB gene, or a Neisseria spp. LOS of immunotype L6 At least one selected from the group consisting of: (b) recovering the Neisseria spp. LOS; (c) hydrazine, alkaline phosphatase, or acyloxyhydrolase Treating the Neisseria genus LOS with a reducing the toxicity of the lipid A moiety. In the method, the order of step (b) and step (c) may be changed. In a preferred method, toxicity in step (c) is reduced to less than 80% of the toxicity of wild type Neisseria LOS as determined in the WEHI assay described above.

  Yet another aspect of the invention relates to a Gram negative OMV (outer membrane vesicle) or blebs comprising a Neisseria LOS as defined above. Means and methods for producing Gram negative OMVs or blebs are described in WO 02/09746. The gram negative bleb thus obtained can be combined with the Neisseria sp. LOS of the present invention to produce a Gram negative bleb containing the Neisseria sp. LOS. Alternatively, the bleb may be produced from a Neisseria strain having the ability to produce the Neisseria LOS of the present invention as defined above. Gram negative blebs containing Neisseria spp. LOS may be further combined with the antigen and / or the pharmaceutically acceptable carrier.

1 Materials and Methods 1.1 Strains Oligosaccharide core mutants and lipid A mutants were obtained from the meningococcal wild strain (WT) H44 / 76 population B described above.

The structure of the oligosaccharide core mutant used in this study is shown in FIG. All strains were grown at 36 ° C. on an aspergillus agar medium (Baging Stoke, UK, manufactured by Difco) supplemented with Vitox (Baging Stoke, UK, manufactured by Oxoid) under an air atmosphere containing 6% CO _2. I let you. The bacteria were used in the stationary phase after 18 hours of culture. Bacteria were suspended in a phenol red-free RPMI 1640 medium (Paisley, UK, manufactured by Gibco), and the absorbance was measured at 540 nm. Absorbance 1 was calculated to correspond to microorganisms 1ml per 10 ^9. Bacteria were fixed in phosphate buffered saline (PBS) containing 0.5% paraformaldehyde (PFA) for 15 minutes and washed thoroughly with RPMI medium. FITC-labeled bacteria were prepared by incubating with 0.5 mg / ml FITC (Pool, UK, Sigma) for 20 minutes at 37 ° C. and then washing thoroughly.

1.2 Cells DCs were prepared from human peripheral blood mononuclear cells (PBMC) as previously described (F. Sallusto and A. Lanzavecchia, 1994, J. Exp. Med. 179: 1109-1118; H Uronen-Hansson et al., 2004, Immunology 111: 173-178). Briefly, monocytes were prepared from PBMC by multi-stage Percoll gradient centrifugation. The fraction of such monocytes was> 95% CD14 + / CD3- / CD19-. 10% heat inactivated FCS, 2.4 mM L-glutamine, 100 U / ml penicillin-streptomycin (all from Paisley, UK, Gibco), 100 ng / ml human recombinant GM-CSF and 50 ng / ml human population Monocytes were incubated for 7 days in RPMI supplemented with IL4 (Hearts, Welwyn Garden City, UK, manufactured by Schering-Plough) to produce DC. The immature DCs prepared in this way were CD14 ^low , CD83- ^ve , CD86 ^low , CD25- ^ve . These CDs also expressed HLA, DR, HLA, DQ, HLA class I, CD40 and CD1a, and were negative for both CD19 and CD3.

In RPMI 1640 supplemented with 10% FCS, DC at a concentration of 5 × 10 ⁵ / ml at a DC / bacterial ratio of 1:50 and 1: 200 (as described in the figure description) was meningococcal H44 / 76. It was cultured with the cells. When measuring intracellular cytokines, 10 μg / mL protein transport inhibitor Brefeldin A (Pool, UK, Sigma) was added.

  Immature DCs were incubated with 20 μg / ml anti-DC-SIGN mAb AZN-D1 for 30 minutes at 37 ° C., followed by incubation with wild type and mutant bacteria for 45 minutes at 37 ° C. The binding specificity for was investigated.

1.3 Binding to DC and internalization Bacterial binding to DC and internalization were examined using a combination of flow cytometry and confocal microscopy. For detection by flow cytometry, DCs were incubated with FITC-labeled bacteria for different times between 30 minutes and 24 hours, fixed on FACSFix (Becton Dickinson), washed and analyzed on a FACS caliber. In a gated DC population, bacteria-bound DC could be easily identified by fluorescence. In confocal microscopy, DC stimulated with FITC-labeled meningococci were placed on an adhesion slide (manufactured by Hertz Bio-Rado Laboratories, UK) for 10 minutes and allowed to adhere. To stop internalization, DC was fixed with 4% PFA for 10 minutes. DCs were visualized by staining with 5 μg / ml anti-MHC class II monoclonal antibody (Grostrap, Denmark, Dako). After washing, the bound antibody was detected with Texas Red conjugate goat anti-mouse antibody (Molecular probes) at 5 μg / ml for 1 hour. The slide was washed and placed in Citifluor (Citifluor, UK). Confocal images were obtained using a Leica SP2 confocal laser scanning microscope system (Leica, Milton Keynes, UK) equipped with an appropriate filter set. In order to identify intracellular bacteria, 15-20 optical sections (0.2-0.5 μm) across the DC were copied and superimposed using Leica confocal imaging software.

1.4 Cytokine measurement To measure intracellular cytokine production, DCs were fixed with PBS containing 4% PFA, 0.1% sodium azide and 0.5% BSA (all manufactured by Sigma) were added. It was washed in the contained PBS, and permeabilized with 25 μl of permeabilizing solution (Caltag). The cells are then incubated for 30 minutes in a dark room at room temperature with an anti-TNF-α monoclonal antibody (Oxford, UK, Becton Dickinson) and IL-12 p40 / 70 (Pharmingen) or isotype matched controls. Incubated. Subsequently, the cells were washed twice in PBS, fixed in Cellfix (CellFix (Becton Dickinson)), and FACS Caliber using Cell Quest Software (Cell Quest Software (Becton Dickinson)). The above was analyzed by flow cytometry. DC constituted a prominent population identified by variance in the forward and right angles. At least 95% of the cells of this population were determined to be DC by expression of MHCII, CD1a, CD25, CD80, CD83 and CD86. Data was collected and analyzed for at least 3000 events at the gate corresponding to DC. Manufacturer of CytoSets ™ ELISA kit (manufactured by Biosource Europe SA, Belgium) for human IL-12, TNF-α, IL-10, IL6 and IL1β for soluble cytokine ELISA assays Used according to the instruction manual.

1.5 Soluble C-type lectin Fc adhesion assay The C-type lectin Fc molecule consists of the extracellular portion of a C-type lectin receptor (DC-SIGN, MGL and Dectin-1B) fused to a human IgG1-Fc fragment at the COOH terminus ( T. Geijtenbeek et al., 2003, J. Exp. Med. 197: 7-17). Soluble C-type lectin Fc adhesion assay was performed as follows. All cells (OD540 = 0.1) were coated on ELISA plates (100 μl / well) for 18 hours at room temperature. Next, 1% BSA was used and blocked at 37 ° C. for 30 minutes. Soluble C-type lectin Fc supernatant was added and placed at room temperature for 2 hours. Unbound C-type lectin Fc was washed away, and binding was measured by an ELIZA method using an anti-IgG1 antibody.

1.6 Differentiation of Th1 / Th2 with DC Add 10% FCS (Berbie, Belgium, Bio Withaker), 500 U / ml IL-4 and 800 U / ml GM-CSF (both from Schering-Plough) In the Iskov modified Dulbecco medium (Gibco), DCs were cultured from monocytes of healthy donors. On day 6, DC maturation was induced with a multiplicity of infection of 10 using a Neisseria meningitidis wild strain and an lgtB mutant. (I) 10 ng / ml E. coli LPS (from St. Louis, MO, Sigma-Aldrich) for mixed Th1 / Th2 reaction assay (ii) 20 μg / ml polyI: C (from Sigma-Aldrich) for Th1 differentiation assay ), And (iii) Th2 differentiation assay, 10 μg / ml PEG2, 10 ng / ml LPS (Sigma-Aldrich) was used as a positive control. On the second day, DC maturation markers (CD80, CD83, CD86 and HLA-DR) were analyzed by flow cytometry. To assess T cell differentiation, mature DCs were incubated with CD45RA ⁺ / CD4 ⁺ T cells (5.10 ³ T cells / 20.10 ³ DC). On days 12-15, resting T cells were restimulated with 10 ng / ml PMA (Sigma-Aldrich) and 1 μg / ml ionomycin (Sigma-Aldrich) for 6 hours. After 1 hour, 10 μg / ml Brefeldin A (Sigma-Aldrich) was added to the T cells. Single cell production of IL-4 and IFN-γ was measured by intracellular flow cytometry analysis. Cells were fixed in 2% PFA, permeabilized with 0.5% saponin (Sigma-Aldrich), anti-human IFN-γ-FITC and anti-human IL-4-PE (Becton, San Diego, CA). Dickinson Pharmigen).

Binding and internalization of meningococcal oligosaccharide core mutants to dendritic cells Using flow cytometry, binding of wild meningococcal strains, oligosaccharide mutants, and LPS-deficient mutants to human DCs And internalization were investigated. Immature DCs are co-cultured with FITC-labeled mutants from Neisseria meningitidis wild strain H44 / 76 at DC / bacterial ratios 1:50 (FIG. 2) and 1: 200 (data not shown) and bind to DCs The presence of the FITC-labeled bacteria was analyzed by FACS. For both wild-type and oligosaccharide mutants, time- and dose-dependent binding to DC was observed. The LPS-deficient mutant showed almost no binding to DC in the first 6 hours of the time course. The lgtB mutant was consistently more binding to DC than the wild or galE mutant at both concentrations.

  Next, in order to distinguish between bacterial binding and internalization, confocal microscopy was used to examine internalization of Neisseria meningitidis wild type and mutant strains into DC (data not shown). FITC bacteria could be easily recognized as small green particles on the slide. Staining with MHC class II was used to visualize DCs. Most DCs had already internalized more lgtB after 1 hour incubation compared to wild type or galE mutant bacteria. Almost no internalization was observed for LPS-deficient mutant bacteria. For lgtB mutant bacteria, it was typical to maintain high surface adhesion over time, in addition to high levels of internalization. These results support the results from FACS, ie, that enhanced bacterial binding to DC results in enhanced bacterial internalization.

Activation and maturation of DC as a response to meningococcal mutants To investigate the relationship between bacterial internalization and cytokine production, IL12 and TNF- produced by dendritic cells in response to wild and mutant strains Intracellular measurement was performed for α, and internal analysis was performed for simultaneous analysis (FIG. 3). DCs were co-cultured with FITC-labeled bacteria at a ratio of 1:50 for 18 hours and then stained to examine intracellular production of TNF-α and IL12. Four quadrants were set in order to distinguish between cells bound by FITC bacteria and cells not bound by FITC bacteria. The percentage is the percentage of DCs that produce intracellular cytokines after bacterial internalization (upper right quadrant) or non-produced DCs (lower right quadrant). Wild strains, lgtB and galE mutants were all strong inducers of TNF-α and IL-12 production by DC, but LPS-deficient mutants were different. Substantially all of the DCs in which the Neisseria meningitidis wild, lgtB, and galE mutants were internalized produced high levels of TNF-α. As for IL-12, production was observed in about 50% of DC in which FITC-labeled bacteria were internalized.

  In addition to measuring intracellular cytokines, the levels of cytokines secreted from DCs in response to meningococci were also measured (FIG. 4). DCs were co-cultured with mutant and wild type bacteria at a ratio of 1: 200 for 18 hours, and the supernatants were collected and analyzed for secreted IL12, IL10, TNF-α, IL6 and IL-1β. The percentage of cytokine production compared to the wild type (wild type = 100%) is shown as a result. Both lgtB and galE mutant bacteria were slightly below wild type bacteria for induction of IL12, TNF-α, and IL-1β. For IL-10 and IL-6, both lgtB and galE were similar to the wild type. In the LPS-deficient strain, cytokine production was hardly observed.

  Finally, the expression of the costimulatory molecules CD40 and CD86 was measured (FIG. 5), and the maturation of DCs stimulated with wild type and mutant bacteria was examined. CD40 and especially CD86 expression was higher in DCs stimulated with lgtB mutant bacteria compared to DCs stimulated with wild type or galE mutant bacteria. In DCs stimulated with lpxA mutant bacteria, almost no expression of CD40 and CD86 was seen.

  In conclusion, these data indicate that binding and internalization of lgtB mutant bacteria results in DC activation and maturation comparable to the level of DC activation and maturation induced by wild-type bacteria. .

Identification of receptors that mediate binding and internalization of lgtB mutant bacteria DC is highly binding and absorbing lgtB mutant bacteria, whether such binding and internalization is mediated by specific DC receptors It became a chance to investigate. Therefore, the present inventors analyzed the ability to bind to a C-type lectin receptor that has oligosaccharide recognition ability and is highly expressed in immature DCs for wild and mutant strains. Binding of a panel of C-type lectin Fc chimera to whole cells of wild-type bacteria and mutant bacteria was examined in a soluble adhesion assay (FIG. 6). The C-type lectin Fc chimera did not bind to wild-type bacteria, galE mutant bacteria, and LPS-deficient mutant bacteria, whereas lgtB mutant bacteria strongly bound DC-SIGN-Fc. Binding of lgtB mutant bacteria to DC-SIGN was also observed in HEK293T cells transiently transfected with K562 cells stably expressing DC-SIGN or DC-SIGN (data not shown). Furthermore, when the phagocytosis of lgtB mutant bacteria by DC was examined in the presence or absence of the anti-DC-SIGN blocking antibody AZN-D1, the specificity of lgtB LPS for DC-SIGN was demonstrated (FIG. 7). In the presence of AZN-D1, lgtB phagocytosis by DC is reduced to the level of binding seen for the wild type, clearly demonstrating that DC-SIGN binds and internalizes lgtB mutant bacteria. It is.

Targeting of lgtB mutant bacteria to DC-SIGN that elicits a T _H 1 immune response If there is a functional relevance to the interaction between lgtB mutant bacteria LPS and DC-SIGN, PFA was evaluated to assess it. After immobilizing either fixed wild-type bacteria or lgtB mutant bacteria to immature DCs, the response of T cells induced by DCs was examined. When DCs pulsed with wild-type bacteria or lgtB mutant bacteria were evaluated by expression of costimulatory molecules and maturation markers, there was no difference in DC maturation (FIG. 8a). However, there was a marked difference in the T _H 1 / T _H 2 profiles induced by these strains (FIG. 8b). In DC pulsed with the wild-type bacterium Neisseria meningitidis, the majority of T cells because they were producing IL-4, was mainly produced T _{H 2} type immune response. lgtB mutant bacteria was the DC primarily pulse, the IFN-.gamma.-producing T cells were induced (evoke), thus the balance of _{T H} 1 cells versus _{T H} 2 cells, was changed to _{T H} 1 cells closer. Such a change in T _H 1 / T _H 2 balance was observed in three different donors. These data indicate that targeting lgtB LPS specifically to DC-SIGN generates a DC signal that induces an immune response to T _H 1, which is highly desirable for adjuvants. It is a phenomenon. See also P. Moingeon et al. (2001) Vaccine 19: 4363-4372.

Binding and internalization of Neisseria meningitidis oligosaccharide / lipid A double mutant to DC Meningococcal wild strain, lgtA and lgtB oligosaccharide mutants, their lpxL1 double mutant, and LPS deficient mutant Binding to human DCs and internalization were examined using flow cytometry. Immature DCs were co-cultured with a FITC-labeled mutant derived from Neisseria meningitidis wild strain H44 / 76 at a DC / bacterial ratio of 1: 100 and analyzed by FACS for the presence of FITC-labeled bacteria bound to DC (Figure 9). The lgtB single mutant was consistently more binding to DC than the wild and lgtA single mutants. Furthermore, the lgtB mutant strain produced higher binding than the wild-type oligosaccharide structure or lgtA oligosaccharide structure even in the lpxL1 mutation background. This result indicates that LPS containing pentaacylated lpxL1 lipid A can enhance lgtB-mediated binding to DC-SIGN.

It is the schematic of the Neisseria meningitidis wild strain L3 LPS and its oligosaccharide mutant. Such a mutant strain can be prepared by inactivation of a gene encoding a glycosyltransferase (indicated by italics). Dendritic cells (DC) were cultured with FITC-labeled bacteria for 24 hours at a 1:50 ratio. Samples were taken at indicated time points to measure bacterial binding to DCs by FACS. Independent representative test results for three different donors are shown. DCs were stimulated at a ratio of 1:50 for 18 hours with FITC-labeled bacteria in the presence of Brefeldin A and stained to examine intracellular TNF-α and IL-12. The dot plot indicates the percentage of DCs that are not producing intracellular cytokines (lower right quadrant) or are (upper right quadrant) for DCs to which bacteria are bound. After 18 hours of co-culture, DC produced TNF-α, IL-12, IL-1β, IL-10 and IL-6 in response to wild-type meningococci and mutants in the culture supernatant. Measured by ELISA. Mean values and SEM values are shown for three independent test results. Expression of CD40 and CD86 after 18 hours co-culture of wild-type meningococci and meningococcal mutants with DC is shown by median fluorescence intensity (MFI). Binding of type C lectin Fc molecules to meningococcal wild and mutant strains in a soluble adhesion assay. Binding of wild meningococcal strains and lgtB mutants to DCs in the presence and absence of 20 μg anti-DC-SIGN antibody AZN-D1 per ml. DC induced Th1 / Th2 cell proliferation. DCs were cultured with meningococcal wild strain H44 / 76, lgtB mutant, E. coli LPS, polyI: C or PEG2 for 48 hours, washed, and (A) maturation marker (average and SEM values) or (B) co-cultured with high purity CD45RA ⁺ CD4 ⁺ T cells, restimulated resting T cells with PMA and ionomycin, and intracellular IL-4 (gray by flow cytometry) ) And IFN-γ (black) were analyzed on a single cell basis. Data from three separate donors are shown. Binding of live bacteria to DC and cytokine induction. Dendritic cells (DC) were cultured at a ratio of 1: 100 with FITC-labeled meningococcal wild strain and LPS mutant. At the indicated times, samples were taken to measure bacterial binding to DCs by FACS. Results are shown for cells from two separate donors.

Claims (11)

Use of Neisseria spp. LOS in the manufacture of a pharmaceutical for immunization of mammals, wherein said Neisseria spp. LOS has the following formula (I)

Wherein LA is a wild-type lipid A moiety or a lipid A moiety having less than 90% toxicity of the wild-type lipid A moiety , GlcNAc is D-glucosamine, Gal is D-galactose, and Glc is D- Glucose, Hep is D-heptose, KDO is 3-deoxy-D-mannooctulosonic acid, PEA is phosphoethanolamine, β1 → 4 is a β-glycoside bond between positions 1 and 4, β1 → 3 Is the β-glycoside bond between the 1 and 3 positions, α1 → 5 is the α-glycoside bond between the 1 and 5 positions, and α1 → 3 is the α between the 1 and 3 positions. A glycoside bond, α1 → 2 is an α-glycoside bond between the 1-position and the 2-position, and α2- → 4 is an α-glycoside bond between the 2-position and the 4-position, and PEA (3 or 6) Phosphoethanolamine is in position 3 or 6 of heptose In may be bound, or at all indicates that may not be present, use and the Neisseria LOS, characterized in that used as an adjuvant. Use according to claim 1, characterized in that Neisseria spp. LOS is used in combination with said antigen in the manufacture of a medicament for enhancing the immune response to an antigen in a mammal. Use according to claim 2, characterized in that the antigen is derived from or produced from bacteria, viruses, fungi, parasites, cancer cells or allergens. 4. Use according to any of claims 1 to 3, characterized in that LA is a lipid A moiety with a toxicity less than 80% of the toxicity of the wild type lipid A moiety as determined in the WEHI assay. The lipid A part is
(A) a lipid A portion obtainable from a Neisseria strain having a mutation that inactivates or inactivates the lpxL1 gene or the lpxL2 gene or a homologue thereof,
(B) a lipid A moiety obtainable by removing one or more secondary O-linked esterified fatty acids from the wild-type lipid A moiety by treatment with an acyloxyhydrolase;
(C) a lipid A moiety obtainable by treating the wild type lipid A moiety with hydrazine, and (d) a lipid A moiety obtainable by dephosphorylation using alkaline phosphatase,
Use according to any of claims 1 to 4, characterized in that it is selected from the group consisting of The lipid A part is
(A) a lipid A moiety having secondary C ₁₂ -acyl only in reduced glucosamine;
(B) a lipid A moiety having secondary C ₁₂ -acyl only on non-reducing glucosamine;
(C) a lipid A moiety lacking any secondary C ₁₂ -acyl group;
(D) a lipid A moiety lacking one or more phosphate groups at either the 1 or 4 ′ position, and (e) deacylation in (a), (b) or (c) above and A lipid A moiety having a combination with dephosphorylation in (d),
Use according to any of claims 1 to 5, characterized in that it is selected from the group consisting of Formula (I)

Wherein L is a wild type lipid A moiety or a lipid A moiety having less than 90% toxicity of the wild type lipid A moiety, and GlcNAc is D- Glucosamine, Gal is D-galactose, Glc is D-glucose, Hep is D-heptose, KDO is 3-deoxy-D-mannococtrosonic acid, PEA is phosphoethanolamine, β1 → 4 is 1st and 4th positions Β-glycoside bond between and β1 → 3 is a β-glycoside bond between positions 1 and 3, α1 → 5 is an α-glycoside bond between positions 1 and 5, α1 → 3 Is the α-glycoside bond between the 1st and 3rd positions, α1 → 2 is the α-glycoside bond between the 1st and 2nd positions, and α2 → 4 is the α-glycoside bond between the 2nd and 4th positions. A glycosidic bond, PEA (3 or 6) is a phosphoeta Indicates that uramin may be bound at position 3 or 6 of heptose, or may not be present at all, and said composition comprises a virus, fungus, parasite, cancer cell, allergen, or Further comprising an antigen derived or produced from a bacterium, wherein the bacterium is Helicobacter, Hemophilus, Bordetella, Chlamydia, Streptococcus, Vibrio, Gram-negative enteric pathogen, Salmonella, Shigella, Campylobacter, Escherichia A composition selected from the group consisting of a genus and a bacterium that causes anthrax, leprosy, tuberculosis, diphtheria, Lyme disease, syphilis, or typhoid fever. 8. The composition of claim 7, further comprising a pharmaceutically acceptable carrier. 9. Composition according to claim 7 or 8, characterized in that the toxicity of the lipid A moiety is less than 80% of the toxicity of the wild type lipid A moiety as determined in the WEHI assay. The lipid A part is
(A) a lipid A portion obtainable from a Neisseria strain having a mutation that inactivates or inactivates the lpxL1 gene or the lpxL2 gene or a homologue thereof,
(B) a lipid A moiety obtainable by removing one or more secondary O-linked esterified fatty acids from the wild-type lipid A moiety by treatment with an acyloxyhydrolase;
(C) a lipid A moiety obtainable by treating the wild type lipid A moiety with hydrazine, and (d) a lipid A moiety obtainable by dephosphorylation using alkaline phosphatase,
The composition according to claim 7, wherein the composition is selected from the group consisting of: The lipid A part is
(A) a lipid A moiety having secondary C ₁₂ -acyl only in reduced glucosamine;
(B) a lipid A moiety having secondary C ₁₂ -acyl only on non-reducing glucosamine;
(C) a lipid A moiety lacking any secondary C ₁₂ -acyl group;
(D) a lipid A moiety lacking one or more phosphate groups at either the 1 or 4 ′ position, and (e) deacylation in (a), (b) or (c) above and A lipid A moiety having a combination with dephosphorylation in (d),
The composition according to claim 7, wherein the composition is selected from the group consisting of:

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